Transgenic aloe plants for production of proteins and related methods

ABSTRACT

The present inventions provide transgenic aloe plants and recombinant constructs for transforming aloe plants, aspects of which, may be applied to other monocots. The recombinant constructs may include one or more DNA sequences encoding mammalian proteins and at least one promoter capable of directing the expression of recombinant proteins in an aloe plant. The present inventions also provide methods for constructing and reproducing a transgenic aloe plant. The present inventions include methods for transfection of an aloe plant with several genes of interest simultaneously. The aloe plant production methods of the inventions may provide the potential to inexpensively and more safely mass-produce some biologically active compounds including biopharmaceuticals for disease therapy, diagnosis and prevention, and is more accessible to the less affluent countries. The aloe plant production methods may also produce proteins for cosmetics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/663,992 filed Oct. 30, 2012, which is a divisional of U.S. patentapplication Ser. No. 13/188,815 filed Jul. 22, 2011, now U.S. Pat. No.8,816,154, which is a divisional of U.S. patent application Ser. No.11/528,056 filed Sep. 26, 2006, now U.S. Pat. No. 8,008,546, whichclaims the priority and benefits of U.S. Provisional Application No.60/720,540 filed Sep. 26, 2005, and which applications are incorporatedherein by reference. A claim of priority to all, to the extentappropriate, is made.

INCORPORATION OF SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web, and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 30, 2012, isnamed SequenceListing.txt.

BACKGROUND OF THE INVENTION

1. Summary of the Invention

The present inventions relate to transgenic monocot plants and, moreparticularly, to transgenic aloe plants and methods and compositions forproducing a transgenic aloe plant and methods for extracting proteinsfrom the transgenic aloe plants.

2. Description of the Related Art

There continues to be a growing market for biologically active proteinsmany of which are used for therapeutic purposes. Currently, there areover 160 protein based medicines available. An additional fifty or soare expected to be approved over the next couple of years. Currentdemand for therapeutic protein production is already outstripping theindustry's capacity. It has been predicted that the industry will needto increase its capacity by four to five times to overcome thisbottleneck. However, production facilities for therapeutic proteins areexpensive and typically take a long time to build. Accordingly, a needexists for production methods that are less expensive and may reduce thetime required to ramp up production.

Some animal based protein production methods are used. However, thesefrequently introduce health risks from diseases. Such risks may arisefrom cross-contamination with diseases that may affect both the animaland the end user such as a human patient. Accordingly, a need exists fora production method that will eliminate the possibility ofcross-contamination between the production organism and the end user.

In addition, many current production methods require extensiveprocessing in order to extract the therapeutic protein from the animalor other host organism in which it was produced and to get the compoundinto a condition where it may be utilized by a patient. Afterpurification, the protein may be combined with an adjuvant or othercarrier material to stabilize the protein and to permit the utilizationby a patient. However, the processes of extraction, purification,resuspension among others involved with the processing of a therapeuticprotein is complex and cumbersome and may not be conducive to use inunderdeveloped countries in need of therapeutics generally. Accordingly,a need exists for simplified production methods which may eliminate orreduce the post extraction processing of therapeutic proteins.

SUMMARY OF THE INVENTION

Compositions and methods in accordance with the present inventions mayresolve many of the needs and shortcomings discussed above and willprovide additional improvements and advantages as will be recognized bythose skilled in the art upon review of the present disclosure.

In one aspect, the present inventions may provide a transgenic aloeplant stably incorporating a gene of interest. In other aspects, novelvectors and constructs may be provided to integrate DNA sequences ofinterest into the genome of an aloe plant. The sequence of interest mayencode for a biologically active protein. The biologically activeprotein may be interferons, immunoglobulins, lymphokines, growthfactors, hormones, blood factors, histocompatability antigens, enzymes,cosmetic proteins and other mammalian proteins, or other proteins ofinterest. In some aspects, the proteins of interest are human proteins.Aloe plants in accordance with the present inventions may transcribe andtranslate the gene of interest into the protein of interest. In oneaspect, at least some of the protein of interest migrates to a centralportion of the aloe leaf. In other aspects, the protein of interest mayinclude a signal sequence to facilitate its translocation into thecentral portion of the aloe leaf. In other aspects, novel methodologiesfor the isolation of individual cells from an aloe plant may beprovided. In still other aspects, the present inventions may providenovel methods for the integration of vectors into an aloe plant andreproduction of such transgenic aloe plants.

Transgenic aloe plants producing mammalian proteins in accordance withthe present inventions may provide a more economically viablealternative for the production of proteins of interest such as forexample various biologically active and cosmetic proteins. In oneaspect, the proteins of interest may be localized and/or concentratedwithin the gel of the aloe leaf. This localization of the proteins ofinterest may simplify the removal of the protein from the plant. In thisaspect, the protein of interest may be co-extracted along with theextraction of the native gel within the central portion of the aloeleaf. Accordingly, the present inventions may provide a transgenic plantfrom which the proteins of interest are generally more readilyaccessible than those from most transgenic plants such as for exampletobacco and corn. Further, the present inventions may provide anefficient method for protein isolation. In still other aspects, theproteins of interest may not be particularly localized within the aloeplant. However, the anatomy and physiology of the aloe plant may stillprovide certain additional advantages for the production of the proteinof interest as will be recognized by those skilled in the art uponreview of the present disclosure.

Aloe plants can offer various advantages over conventional methods forproducing proteins of interest in bacteria and yeast. The advantages ofaloe plants may include the ability to process proteins in ways that thesimple single cell bacteria and yeast are poorly suited and which may benecessary to produce the proteins in the desired form. This processingcan include the chemical modification, such as by glycosylation, andfolding of some proteins for example. Further, in comparison to otherprotein production methods based on animal cells, aloe plant productionmay offer significant cost benefits, scalability advantages and areduced risk of contamination that may be harmful to humans.

A protein modified gel from the central portion of the aloe leaf of atransgenic aloe plant in accordance with the present inventions may beused directly from the aloe plant. This may avoid the need forrelatively complex and expensive extraction of the proteins of interestfrom native plant materials. In some aspects, the gel extracted from theleaf may be used directly without the need for protein extraction orprocessing. The gel may be in the form of the pith mechanicallyextracted from an open end of a broken leaf of a transgenic aloe plant.

The present inventions may provide economically viable alternatives forthe production of human and other mammalian proteins which arebiologically active and/or have cosmetic applications.

Upon review of the present disclosure, those skilled in the art willrecognize additional improvements and advantages of the presentinventions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example of the cross-sectional anatomy of an aloeleaf;

FIG. 2 illustrates exemplary methods for generation of callus tissue;

FIG. 3 diagrammatically outlines various steps for generating atransgenic aloe plant;

FIG. 4 diagrammatically illustrates components of a plasmid vectorincluding sequence information (DNA sequence disclosed as SEQ ID NO: 45and protein sequence disclosed as SEQ ID NO: 46);

FIG. 5 lists the sequence information for the ubiquitin promoter frommaize and highlights regions of the ubiquitin promoter (SEQ ID NO: 44);

FIGS. 6A and 6B illustrate the construction of a plasmid vector systemin accordance with aspects of the present inventions;

FIGS. 7A to 7C illustrate the construction of another plasmid vectorsystem in accordance with aspects of the present inventions;

FIG. 8 illustrates the construction of additional plasmids vector systemin accordance with aspects of the present inventions; and

FIG. 9 illustrates the construction of integration systems in accordancewith aspects of the present inventions

All Figures are illustrated for ease of explanation of the basicteachings of the present inventions only; the extensions of the Figureswith respect to number, position, sequence, relationship andcompositions of the various embodiments will be explained or will bewithin the skill of the art after review of the following description.Further, the various protocols, tools, and compositions to practice todisclosed inventions will be within the skill of the art after review ofthe following description.

DETAILED DESCRIPTION OF THE INVENTIONS

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

As used herein a “transformed aloe cell” means a plant cell that istransformed with stably-integrated, non-natural, recombinant DNA, e.g.by Agrobacterium-mediated transformation or by bombardment usingmicroparticles coated with recombinant DNA or other means. A transformedAloe cell of this inventions can be an originally-transformed plant cellthat exists as a microorganism or as a progeny plant cell that isregenerated into differentiated tissue, e.g. into a transgenic Aloeplant 10 with stably-integrated, non-natural recombinant DNA, or seed orpollen derived from a progeny transgenic Aloe plant 10.

As used herein a “transgenic aloe plant ” means an aloe plant whosegenome has been altered by the stable integration of recombinant DNA. Atransgenic aloe plant 10 includes an aloe plant regenerated from anoriginally-transformed aloe cell and progeny transgenic aloe plants fromlater generations or crosses of a transformed aloe plant 10.

As used herein “recombinant DNA” means DNA which has been a geneticallyengineered and constructed outside of a cell including DNA containingnaturally occurring DNA, cDNA, synthetic DNA and/or other DNA.

As used herein “promoter” means regulatory DNA for initializingtranscription. A “plant promoter” is a promoter capable of initiatingtranscription in aloe cells whether or not its origin is a aloe cell,e.g. is it well known that Agrobacterium promoters are functional inaloe cells. Thus, plant promoters include promoter DNA obtained fromplants, plant viruses and bacteria such as Agrobacterium andBradyrhizobium bacteria. Examples of promoters under developmentalcontrol include promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, or seeds. Such promoters arereferred to as “tissue preferred”. Promoters that initiate transcriptiononly in certain tissues are referred to as “tissue specific”. A “celltype” specific promoter primarily drives expression in certain celltypes in one or more organs, for example, vascular cells in roots orleaves. An “inducible” or “repressible” promoter is a promoter which isunder environmental control. Examples of environmental conditions thatmay effect transcription by inducible promoters include anaerobicconditions, or certain chemicals, or the presence of light. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most conditions. Thepromoter may include enhancers or other elements which affect theinitiation of transcription, the beginning site of transcription, levelsof transcription, the ending site of transcription, or anypostprocessing of the resulting ribonucleic acid.

The term “genetic construct” as used herein is defined as a DNA sequencecomprising a synthetic arrangement of at least two DNA segments for thepurpose of creating a transgenic aloe plant. In a specific embodiment,one segment is a regulatory sequence and another segment encodes a geneproduct.

As used herein “operably linked” means the association of two or moreDNA fragments in a DNA construct so that the function of one, e.g.protein-encoding DNA, is controlled by the other, e.g. a promoter,termination sequence, etc.

The term “transcription” as used herein is defined as the generation ofan RNA molecule from a DNA template.

The term “translation” as used herein is defined as the generation of apolypeptide from an RNA template.

As used herein “expressed” means produced, e.g. a protein is expressedin a plant cell when its cognate DNA is transcribed to mRNA that istranslated to the protein.

The present inventions may provide novel transgenic aloe plants 10expressing various proteins of interest, may provide methods andcompositions for producing transgenic aloe plants 10, may providemethods for extracting proteins from the transgenic aloe plants 10, andmay provides novel compositions of proteins of interest and componentsfrom the transgenic aloe plant 10. The proteins of interest produced inaccordance with the present inventions may include a secretory signal tofacilitate their accumulation in the pith 18 or pith of an aloe leaf 12.In one aspect, this accumulation may occur, at least in part, within themusalegenous cells of the leaf of a transgenic aloe plant 10.

The pith 18 or pulp of an aloe leaf 12 include the components of theleaf which, at least in part, are commonly referred to as the “gel” whenextracted from the aloe leaf 12. As used herein, “gel” will refer to theextracted pith 18 and other associated materials which accompany thepith 18 as it is extracted from the aloe leaf 12 regardless of thedegree of subsequent processing. In accordance with one or more aspectsof the present inventions, the gel from the aloe leaf 12 may have amodified composition. In one particular aspect, the composition of thegel may be modified to include at least one exogenous protein componentto be referred to as a “protein modified gel.” Extraction of a proteinmodified gel with the associated protein(s) of interest may offerreadily accessible proteins and/or efficient methods for proteinisolation. The protein modified gel produced and/or extracted inaccordance with the present inventions may be used directly without theneed for protein extraction.

Aspects of the present inventions are generally illustrated in FIGS. 1to 8 for exemplary purposes. The present inventions provide transgenicplants, compositions and methodologies that are generally applicable tothe genus aloe of the family Liliacae. Typically, the present inventionsare described with reference to application in the species is selectedfrom the group of Aloe vera (barbadensis miller), Aloe ferox and Aloearborescence for exemplary purposes. The Genus Aloe generally includes agroup of large stemless rosette succulent monocot plants. These plantsare generally referred to as aloe plants 10 for purposes of the presentdisclosure.

Various compounds produced by aloe plants have been used for medicinalpurposes. These compounds when present in a protein modified gel maycomplement the medicinal properties of biologically active proteins of atransgenic aloe plant 10 in accordance with the present inventions. FIG.1 illustrates a cross section through a leaf 12 of a transgenic aloeplant 10. The leaves of the transgenic aloe plant 10 include an easilyextractable gelatinous mixture of proteins, carbohydrates and waterincluded in the gel which is primarily derived from the pith 18. Thisgelatinous mixture is primarily located in the central portion of analoe leaf 12. Various compounds in the gel have been shown to have anumber of medicinal properties and uses. In one aspect, the gel and/orpith 18 stabilize a transgenic protein produced by a transgenic aloeplant 10 and localized in the gel and/or pith 18.

FIG. 1 particularly illustrates the epidermis 14, the cortex ormesophyll 16, and the pith or pulp 18. The epidermis 14 forms the outerlayer of cells of the leaf In one aspect of the present inventions, atransgenic aloe plant may include aloe cells in one or more of thesetissues which transiently or stably incorporate a genetic constructwhich is expressed by the aloe cell. The cortex 16 includes cells richin chloroplasts as well as the vascular bundles, xylem and phloem. Thepith 18 is the spongy parenchyma composed almost exclusively of largecortex cells and, at least in part, represents the gel where it may beadvantageous to incorporate or accumulate one or more transgenicproteins of interest.

The epidermis 14 typically consists of a single outer layer of cells.Just beneath the epidermis 14 is the cortex 16 including the network ofvascular bundles. The outer support of the vascular bundles is generallyprovided by the sheath cells. The vascular bundles are composed of threegeneral types of tubular structures: the xylem, the phloem, and theassociated large pericyclic tubules. The xylem transports water andminerals from the roots to the leaf of the plant. The phloem transportsstarches and other synthesized materials throughout the plant. Thepericyclic tubules contain a latex or sap which is very high in thelaxative anthraquinones, especially aloin. The anthraquinones absorbultra violet rays of the sun and prevent overheating of the centralportion of the Aloe leaf 12 which generally functions as the waterstorage organ of the aloe plant. The pericyclic portion of the vascularbundles are adherent to the epidermis 14, while the remainder of thevascular bundles protrude into the pith 18. The innermost and majorportion of the aloe leaf 12 is the pith 18 which, at least in part,constitutes the gel. For purposes of gel extraction, the epidermis 14and cortex 16 may be generally considered to comprise the sheath whichcontain the gel. The extracted gel, comprised substantially of the pith18, is typically thick and slimy substance that has been historicallybeen topically applied to skin for medicinal purposes, such as forexample as a therapy for burns and wounds.

The gel generally functions as a reservoir of materials for the aloeplant. The cortex 16 typically synthesizes many of the carbohydrates andglycoproteins which are needed by the aloe plant. Carbohydratessynthesized in excess of are typically transported to the pith 18 forstorage along with water and some minerals. The carbohydrates aretransported by the phloem vessels to large vacuoles within the cortex 16cells of the pith 18. Water is then osmotically attracted to thecarbohydrates permitting the pith 18 to function the water storage organof the aloe plant.

The gel is relatively easily extracted by breaking an aloe leaf 12 alongits longitudinal axis and crushing the leaf to force the gel from thesurrounding sheath through the break. Additional materials may extractedfrom the tissues of the leaf along with the gel.

Process Overview

Transgenic aloe plants 10 in accordance with one or more of the presentinventions generally include one or more DNA constructs stablyincorporated within the plant to express one or more proteins ofinterest. Generating a transgenic aloe plant 10 in accordance with oneor more of the present inventions may involve a variety of novelcompositions and methods. Typically, one or more DNA constructs aredeveloped to express one or more proteins in the transgenic aloe plant10. In one aspect, a single construct may express a single mRNA. Inother aspects, a single construct may express multiple mRNA. In stillother aspects, multiple constructs may produce multiple mRNA. Each mRNAmay produce one or more polypeptides.

To introduce the constructs into the aloe plants, aloe cells orundifferentiated callus tissue is typically used as generallyillustrated in FIG. 2. The aloe cells and callus tissue are typicallyderived from the root or leaf meristem tissues or from a seed of an aloeplant. The DNA constructs are introduced into the aloe cells using arange of techniques and/or constructs as is diagrammatically illustratedin FIG. 3. Some techniques will be recognized by those skilled in theart upon review of the present disclosure. Aloe cells or callus tissueincorporating the desired constructs are selected for using varioustechniques. Typically, the DNA construct will include a selectablemarker. Once the desired construct has been stably introduced into thealoe cells or callus tissue, aloe plantlets are typically generated fromthe aloe cells or callus tissue. The aloe plantlets that develop intoviable transgenic aloe plants 10 containing the DNA constructs mayeither constitutively produce or inducibly produce the desiredprotein(s) of interest. The protein of interest may be localized in thetransgenic aloe plant 10 or may be found generally throughout thetransgenic aloe plant 10 depending on the particular protein beingproduced and/or the presence or absence of a signal sequence associatedwith the protein. Depending upon the particular construct and/orassociated proteins of interest, the transgenic aloe plant 10 may thenbe vegetatively or sexually propogated. The proteins may be isolatedfrom the transgenic aloe plants 10 using a wide range of techniques. Theproteins may then be further isolated and/or further processed. Suchprocessing may include enzymatic modification, chemical modification,incorporation into a suitable adjuvant, among other processing that willbe recognized by those skilled in the art upon review of the presentdisclosure. In one aspect, the proteins may be processed from aninactive (precursor) into active form for specific applications of theparticular protein.

Isolation of Cells

As shown in the exemplary sequence illustrated in FIG. 2, aloe cells orgroups of aloe cells are typically isolated from an aloe plant prior tothe incorporation of the desired construct. The types of aloe cellgenerally chosen for incorporation of DNA constructs are generallychosen based on their regenerative potential. Meristematic cells fromthe shoot meristem or the root meristem from an aloe plant or embryonicaloe cells from an aloe seed may be used. However, many parts of thealoe plant retain the potential to regrow or form callus tissue and mayalso be utilized. The cells are typically isolated using a range oftechniques that will be recognized by those skilled in the art uponreview of the present disclosure. Typically, the cells are mechanicallyisolated from the aloe plant using a scalpel. Alternatively, othertechniques mechanical or otherwise may be used to isolate the necessarycells as will be recognized by those skilled in the art upon review ofthe present disclosure. Once an appropriate aloe cell or group of aloecells is isolated, the aloe cells are typically grown in culture to formcallus tissue. Although certain techniques may not require that the aloecells are first grown into callus tissue, the callus tissue provides asource of undifferentiated set of aloe cells retaining the potential forgenerating a transgenic aloe plant 10. The callus tissue is typicallygrown on a solid medium. However, the callus tissue can also be place ina liquid culture medium and grown in suspension.

The meristematic cells may be isolated from the tips of the roots orleaf of an aloe plant. Meristematic aloe cells may also be isolated fromthe apical meristem. Typically, the aloe plant from which the tissuesare isolated is a young healthy aloe plant. The aloe plant is typicallyselected to be no larger than eight (8) inches with six (6) or fewerprimary shoots. A wound is typically formed on a shoot of an aloe plantby cutting it into segments. The wounded surface may encourage the aloecells to grow. It is typically primarily on the surface of the cuttingthat the callus will begin to grow. To isolate aloe cells from an aloeleaf 12, segments are typically cut from the distal end of a younggrowing shoot. Portions of the segment are then plated on a growthmedium. Shoot apical meristem tissue is derived from a one inch cuttingfrom the base of a young aloe plant. The aloe leaves 12 are removed fromthe cutting and the segment is plated. The meristem aloe cells are foundwithin the “cutting”. They are “selected for” or “isolated” by theirability to continue growing on the culture plate—forming callus tissueor regenerating shoots. The meristematic aloe cells once isolated arethen cultured on appropriate medium and under conditions promoting theformation of callus tissue.

The isolation of embryonic aloe cells from seeds generally involvesremoval of the seed coat to expose the embryo and the mechanical removalof the desired aloe cells from the embryo. The aloe seeds are typicallysterilized and the outer husks of the seed are removed. To mechanicallyremove the aloe cells from either a aloe plant or an aloe embryo, ascalpel is typically used. Embryonic aloe cells, once isolated from theseed coat are then cultured on appropriate medium and under conditionspromoting the formation of callus tissue.

When callus tissue is desired, the isolated aloe cells are typicallygrown under conditions favoring the formation of callus tissue as willbe recognized by those skilled in the art upon review of the presentdisclosure. Typically, the isolated aloe cells are plated and grown inan appropriate solid nutrient medium to form callus tissue having a sizeof roughly 1 cm in diameter prior to transformation. Aloe cells aretypically grown between 23 and 26 degrees Celsius. Suitable mediumsinclude a base solid or liquid which will typically include supplementalinorganic nutrients—both macroelements (such as nitrogen, sulphur,phosphorus, calcium, magnesium, and potassium, and microelements (suchas iron, boron, cobalt, copper, iodine, manganese, molybdenum, andzinc); organic nutrients including sugars (sucrose or maltose) andvitamins and cofactors (thiamine, niacin, biotin,pyridoxine,myo-inositol among others); amino acids (such as proline and caseinhydrolysate); as well as growth regulators primarily a source of auxin(typically (NAA), 1-naphthaleneacetic acid, (IAA), indole-3-acetic acid,or (2,4-D), 2,4-dichlorophenoxyacetic acid) and a source of cytokinin(typically (BAP) 6-benzylaminopurine). These mediums and vitamins aretypically commercially available in pre-formulated compositions withvarying concentrations of inorganic nutrients and vitamins and areknown, among others, as MS media (Murashige-Skoog), Gamborg media, orChu N6 media. Additionally, cells grown on Petri dishes typicallyrequire a solid matrix support such as agar to be added to the growthmedia.

Formation of DNA Constructs

Suitable DNA constructs are typically introduced into callus tissuederived from isolated aloe cells to allow the production of theprotein(s) of interest by the resulting transgenic aloe plant 10. DNAconstructs may also be introduced into isolated aloe cells or maturealoe plants as may be recognized by those skilled in the art upon reviewof the present disclosure. DNA constructs are typically incorporatedinto a vector such as a plasmid or virus for propagation of theconstruct and for introduction into the aloe cells. An exemplary plasmidvector may include the plasmid marketed under the tradename pZErO byInvitrogen (Carlsbad, Calif.), diagrammatically illustrated in FIG. 4for exemplary purposes, and may include one or more of the functionalunits, for example, of this plasmid.

DNA constructs in accordance with the present inventions can include apromoter sequence capable of functioning in an aloe cell, a sequenceencoding a protein of interest, a terminator sequence, and atranslational start and stop site all of which are capable offunctioning in an aloe cell. These components when properly configuredand combined can initiate transcription of DNA and translation of mRNAand their respective termination in an aloe cell. The DNA construct mayalso include a secretion signal. Alternatively, some proteins ofinterest, such as for example interferon, may include a domain, naturalor synthetic, that targets interferon for secretion. The secretionsignal is typically cleaved as the protein leaves the cell or, in somecases, may be retained by the protein without substantially affectingthe protein's function. Secretory signals may be added to a variety ofproteins that may require secretory signals for translocation. Thesesecretory signals may be derived from various plant secretion sequences.The DNA construct or an associated vector may also include at least oneselectable marker. In one aspect, the DNA construct may include both afirst selectable marker for propagation of the vector in bacteria and aselectable marker for growth in aloe cells. In addition, the DNAconstruct may include other regulatory elements as well for expressionin specific aloe cells in the aloe plant 10. Other construct componentsmay include additional regulatory elements, such as 5′ leaders andintrons for enhancing transcription, 3′ untranslated regions (such aspolyadenylation signals and sites), DNA for transit peptides.Amplification of a desired DNA sequence, such as a sequence encoding adesired therapeutic protein, may be accomplished by the polymerase chainreaction. (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporatedherein by reference). Upon review of the present disclosure, thoseskilled in the art will recognize that the DNA constructs, including thepromoter sequences, the regulatory sequences, the stabilizing sequences,the targeting sequences and/or the termination sequences may be modifiedto affect their function using methods known to those skilled in theart.

As noted above, a promoter that is operable in an aloe cell is typicallyutilized. The promoter typically contains genetic elements at whichregulatory proteins and molecules may bind. These proteins typicallyinclude RNA polymerase and other transcription factors. The promoter isoperatively linked in a functional location and/or orientation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence. The promoter may or may not be used inconjunction with an “enhancer,” which refers to a cis-acting regulatorysequence involved in the transcriptional activation of a nucleic acidsequence.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in at leastone cell type of an Aloe plant 10 in which expression is desired. Thoseof skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,for example, see Sambrook et al. (1989), incorporated herein byreference. The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous. The DNA constructstypically require transcriptional and translational initiation andtermination regulatory signals capable of functioning in aloe cells. Alarge variety of sequences regulating transcriptional initiation may beused. DNA sequences controlling transcription initiation may come fromAgrobacterium, viruses or plants. The 35S viral transcription initiationregion from cauliflower mosaic virus (35S-CaMV) may be used for aloeplants 10. Plant promoters which may be used in aloe plants 10 may alsoinclude the ribulose-1,5-bisphosphate carboxylase (RUBISCO) smallsubunit promoter from various monocot or dicot plants or the ubiquitinpromoter from maize. Other suitable promoters may be used and may berecognized by those skilled in the art upon review of the presentdisclosure. If inducible regulation is desired, domains may be obtainedfrom different sources so that a regulatory region from one source iscombined with an RNA polymerase binding domain from another source.Regulation of expression may be to a particular stage of a transgenicaloe plant's 10 development in a specific part of the transgenic aloeplant 10 like roots, leaves, seeds, flowers, sap or in a combination ofplant parts and developmental stages. Regulation of expression to aparticular stage of development or tissue may require additional DNAelements as will be recognized by those skilled in the art upon reviewof the present disclosure.

Numerous other promoters that are active in plant cells have beendescribed in the literature and may be usable in aloe cells. Theseinclude promoters present in plant genomes as well as promoters fromother sources, including: nopaline synthase (NOS) promoters and octopinesynthase (OCS) promoters carried on tumor-inducing plasmids ofAgrobacterium tumefaciens; and caulimovirus promoters such as thecauliflower mosaic virus. In addition, various other promoters have alsobeen identified in various references, including, but not limited to,U.S. Pat. Nos. 5,858,742 and 5,322,938, which disclose versions of theconstitutive promoter derived from cauliflower mosaic virus (CaMV35S);U.S. Pat. No. 5,641,876, which discloses a rice actin promoter; U.S.Patent Application Publication 2002/0192813A1 which discloses 5′,3′ andintron elements useful in the design of effective plant expressionvectors; U.S. patent application Ser. No. 09/757,089, which discloses amaize chloroplast aldolase promoter; U.S. patent application Ser. No.08/706,946, which discloses a rice glutelin promoter; U.S. patentapplication Ser. No. 09/757,089, which discloses a maize aldolase (FDA)promoter; and U.S. patent application Ser. No.60/310,370, whichdiscloses a maize nicotianamine synthase promoter, all of which areincorporated herein by reference. These and numerous other promotersthat function in plant cells and may be operable in a transgenic aloeplant for expression of desired therapeutic proteins.

As one particular example, the ubiquitin promoter (SEQ. ID. NO. 44) frommaize may be used. The ubiquitin promoter from maize is a large element,almost 2 kb in length, composed of at least three general regions. Thesequence of which is listed in FIG. 5 with the particularly relevantfeatures labeled. The first section of the ubiquitin promoter is locatedat the most 5′ end contains matrix attachment regions (MARs) which areelements that interact with histones and other nuclear proteins andserve to “loop out” flanking sequences making them more readilyaccessible to the cell's transcriptional machinery. They also help toinsulate transcriptional units from one another, which is important inpreventing transcription initiated in one place from “reading through”into a second sequence. This may help reduce the risk of creatingantisense messages.

The second section contains enhancer elements and the actual promoter.The enhancer elements bind transcription factors that are responsiblefor directing the transcriptional machinery, the pol II complex, to bindto the promoter and initiate transcription. While the “promoter”specifically refers to the essential DNA elements necessary to interactwith the core transcription initiation machinery, the term promoter ismore generally used to encompass all of the DNA elements involved withtranscription initiation, and in this case the “ubiquitin promoter” isloosely used to mean all of the 2 kb region or modifications of, 5′ tothe cloned gene of interest.

The third section contains an approximately 1 kb intron. Introns areregions of a transcribed gene that are removed from the final translatedmessage, the mRNA, through the process of splicing. Introns have alsobeen found to influence gene expression directly, by providingalternative enhancer elements, as well as by increasing proteintranslation by facilitating the translocation of RNA messages from thenucleus to the cytoplasm—a process linked in part to splicing. However,the presence of introns in transgenic systems does not always lead toincreases in RNA expression or levels of translated protein. In certainaspects, the ubiquitin intron may be modified to reduce the overall sizeof the vectors and to assess their effect on transgenic gene expressionin the aloe.

In other aspects of the present inventions, it may be desired forpreferential expression in green tissues of the aloe plant. Promoters ofinterest for such uses may include those from genes such as Arabidopsisthaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit(Fischhoff et al. (1992) Plant Mol Biol. 20:81-93), aldolase andpyruvate orthophosphate dikinase (PPDK) (Taniguchi et al. (2000) PlantCell Physiol. 41(1):42-48).

As noted above, the promoters may be altered to contain multiple“enhancer sequences” to assist in elevating gene expression. Suchenhancers are known in the art. By including an enhancer sequence withsuch constructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedupstream (5′) or downstream (3′) to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Particularly usefulas enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No.5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase geneintron, the maize heat shock protein 70 gene intron (U.S. Pat. No.5,593,874) and the maize shrunken 1 gene.

Constructs in accordance with aspects of the present inventions mayinclude a 3′ element that typically contains a polyadenylation signaland site. Well-known 3′ elements include those from Agrobacteriumtumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr73′, for example disclosed in U.S. Pat. No. 6,090,627, incorporatedherein by reference; 3′ elements from plant genes such as wheat(Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitingene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene arice lactate dehydrogenase gene and a rice beta-tubulin gene, all ofwhich are disclosed in U.S. published patent application 2002/0192813A1, incorporated herein by reference; and the pea (Pisum sativum)ribulose biphosphate carboxylase gene (rbs 3′), and 3′ elements from thegenes within the host plant.

A protein of interest may be encoded by a structural gene incorporatedinto the DNA construct. The structural gene may be a mammalian gene orportions of a mammalian gene. Structural genes of interest may encodefor interferons, immunoglobulins, lymphokines, growth factors, hormones,blood factors, histocompatability antigens, enzymes, or other proteins.The sequence for interferon alpha 2 is listed for exemplary purposes asSEQ. ID. NO. 33. Structural genes may also encode markers proteins likethe green fluorescent protein (GFP) from jellyfish. The DNA sequence ofthe structural genes may be modified to allow high level expression inan aloe plant 10. The codon bias for the aloe plant 10 may differ fromthe codon bias in the original species from which the structural genewas isolated. The native structural gene may be engineered to optimizethe production of the encoded protein in aloe plant 10 s. Further, theDNA sequence of the structural gene may be engineered to provide forappropriate glycosylation in aloe plants 10that does not interfere withthe structure of the protein.

Termination of transcription and translation may be provided by avariety of transcriptional and translational termination sequences whichare capable of functioning in an aloe plant 10. In one aspect, thetermination sequences may include the sequence from the nopalinesynthetase (NOS) gene from Agrobacterium. In other aspect, thetermination sequence may be derived from the termination sequences ofnative aloe genes and proteins.

A marker gene will often be integrated into the DNA construct. Themarker gene may allow cells that contain the structural gene of interestto be selected from the population of all cells which do not contain themarker gene. Marker genes may include enzymes or other proteinsproviding resistance to kanamycin, chloramphenicol, G418 and gentamycinand among others. Still other specific DNA sequences may be necessary iffor example Agrobacterium is used as a vector. Other regions may bepresent if the DNA is to be targeted to a specific cell type.

As referenced above, the DNA constructs may also include a secretionsignal. Constructs may also include a translocation sequence encoding asignal peptide which may target the therapeutic protein for removal fromthe cell in which the protein was formed. Various signal sequences havebeen identified in the literature that are functional in plants and,more particularly, functional in monocot plants such as aloe plants.These may be operably integrated into the constructs or within the genesof interest. In one aspect, the alpha amylase secretory sequence (SEQ.ID NO. 29) from rice (Oryza sativa) may be utilized. This signalsequence is characterized in “The alpha-amylase genes in Oryza sativa”Mol Gen Genet., 1990 April; 221(2):235-44, the disclosure of which ishereby incorporated by reference in its entirety. Alternatively, somegenes of interest, such as for example interferon, may include a domain,natural or synthetic, that targets interferon for secretion. Thesecretion signal is typically cleaved as the protein leaves the cell or,in some cases, may be retained by the protein without substantiallyaffecting the protein's function. Secretory signals may be added to avariety of proteins that may require secretory signals fortranslocation. These secretory sequences may be derived from variousplant secretion sequences.

FIGS. 6A to 6B illustrate an exemplary set of constructs for producing aprotein of interest from an Aloe plant. The plasmid constructions arelabeled as pzUI (TGC9). TGC9 includes the full length ubiquitin promoterdriving expression of human interferon alpha 2, and pzUEK (TGC12) (SEQ.ID. NO. 38), the full length ubiquitin promoter driving expression of afusion protein containing eGFP (enhanced green fluorescent protein)(SEQ. ID. NO. 31) and the kanamycin resistance protein. The fusionprotein combines the screening properties of both proteins (selectionand visualization) in one.

As illustrated, the plasmid pzI (TGC8) was created by PCR amplificationof the human interferon alpha 2 gene using PCR primers that each containtwo distinct restriction enzyme sites (see Table I for primer sequence).The amplified interferon (IFN) gene has flanking 5′ PstI and BamHI sitesand 3′ Sad and XhoI sites. The PCR generated IFN was then cloned as aPstI ahoI fragment into the pZErO vector to create the construct pzI(TGC8) and introduce the BamHI and Sad sites for subsequent cloning.This construct contains the full-length IFN alpha gene and its signalsequence, responsible for directing the secretion of the IFN proteinfrom the cell. This is depicted on Slide 3. These PCR generated cloneswere sequenced to confirm the absence of any DNA mutations.

As illustrated, the plasmid vector pzUI (TGC9) was created by cloningthe IFN alpha 2 gene downstream of the full-length ubiquitin promoter.The PstI/XhoI fragment of IFN was released from pzl (TGC8) and ligatedinto pzU (TGC1) to create the construct pzUI (TGC9). This is depicted onSlide 3.

As illustrated, the eGFP gene was PCR amplified using a set of primerscontaining BamHI (5′end) and XhoI (3′end) restriction sites (see Table Ifor primer sequence). This PCR generated eGFP gene was created without astop codon (to ultimately allow for expression of an eGFP-kan fusionprotein). Thus translation does not terminate at the end of the eGFPsequence. The PCR amplified fragment was digested with restrictionenzymes BamHI and XhoI and ligated into the BamHI and XhoI sites ofpZErO, creating pzEnostop (TGC10). This is depicted on Slide 3. PCRgenerated clones were sequenced to confirm the absence of any DNAmutations.

As illustrated, pzEK (TGC11) generates a fusion construct between theeGFP gene and the kanamycin resistant gene. eGFP was released as aBamHI/XhoI fragment from pzEnostop (TGC 10) and ligated into pzK (TGC4)to create the construct pzEK (TGC11). The eGFP gene is cloned in frameand 5′ to kan/nos (SEQ. ID. NO. 35).

As illustrated, a BamHI/XbaI fragment from pzEK (TGC 11) containing theeGFP/kan/nos cassette, was cloned into the BamHI and XbaI sites of pzUI(TGC9) in order to allow for expression of the eGFP/kan fusion gene. TheIFN sequence is thus removed. This new construct, pzUEK (TGC12),expresses the eGFP/kan fusion protein from the full-length ubiquitinpromoter (depicted in slide 4). This fusion protein retains thecharacteristics of each of the individual proteins, and thus has theobvious benefit of functioning both as visual marker and selective agentin one.

FIGS. 7A to 7C illustrate another exemplary set of constructs forproducing a protein of interest from an Aloe plant. The illustratedvectors allow for the expression of two genes, as a single transcript,while still being translated into two distinct proteins. To accomplishthis, the two genes are separated from one another by an interveningIRES element (internal ribosome entry sequence) (SEQ. ID. NO. 34). TheIRES element provides a second, internal site, for ribosome attachmentand translation. This second site allows for transcripts cloned 3′ tothe IRES element, to be separately translated. These vectors, namelypzUSEIrK (TGC18) (SEQ. ID. NO. 43) and pzUIIrK (TGC19) (SEQ. ID. NO.39), were constructed in stages, ultimately allowing for the expressionof eGFP or IFN (respectively) together with the kanamycin resistancemarker.

As illustrated, the pzEnostart (TGC13) vector was created by firstamplifying eGFP by PCR (table I for primer sequences) using primerscontaining the restriction enzyme sites BamHI (5′end) and Sad (3′end).This PCR product was cloned as a BamHI/SacI fragment into pZErO. TheeGFP gene in this construct lacks an ATG translation start site, as thisprotein is expressed as a fusion with the alpha amylase signal sequence(see construction of pzSE, TGC14). This is depicted on Slide 5. ThesePCR generated clones were sequenced to confirm the absence of any DNAmutations.

As illustrated, the one strand of the signal sequence from the alphaamylase gene was synthesized and then made double stranded by fill inusing PCR (table I for primer sequences). The signal sequence was thendigested with the restriction enzymes PstI and BamHI and cloned intopzEnostart (TGC13). This is depicted on Slide 5. The signal sequence wascloned in frame, 5′ to eGFP, and provides the translation initiationsite for signal sequence eGFP fusion. Resulting pzSE (TGC14) clones weresequenced to confirm the absence of any DNA mutations.

As illustrated, the vector pzUSE (TGC15) was constructed to link thealpha amylase signal sequence (ss)-eGFP fusion with the ubiquitinpromoter. The gene cassette containing the ss-eGFP from pzSE (TGC14) wascloned as a PstI/SacI fragment into pzUI (TGC9). This replaces the IFNgene cassette (released from pzUI (TGC9) by digesting with restrictionenzymes PstI and SacI) creating pzUSE (TGC15). This is depicted on Slide5.

As illustrated, the IRES element was amplified by PCR (table I forprimer sequences) from pIRES2-EGFP (Clontech, BD Biosciences) withprimers containing the restriction sites SacI (5′end) and XhoI (3′end).The amplified IRES element was cloned as a SacI/XhoI fragment into pZErOto create pzIr (TGC16). This is depicted on Slide 6. These PCR generatedclones were sequenced to confirm the absence of any DNA mutations.

As illustrated, the IRES element from pzIr (TGC16) was then cloned as aSacI/XhoI fragment into pzK (TGC4) to create pzIrK (TGC17). Thuspositioning the IRES upstream of the kan/nos gene cassette.

As illustrated, the IRES-kan/nos gene cassette was cloned as aSacI/IXbaI fragment into pzUSE (TGC15) to create pzUSEIrK (TGC18). Thisis depicted on Slide 6. This construct expresses eGFP as a fusion withthe alpha amylase signal sequence, targeting the eGFP for secretion fromthe cell. This makes it possible to visually monitor protein traffickingand accumulation within the transformed plant. This vector alsoexpresses the kanamycin resistance protein (translated from the internalIRES element) and allows for selection in transgenic plants.

As illustrated, the IRES-kan/nos gene cassette was also cloned as aSacI/XbaI fragment into pzUI (TGC9) to create pzUIIrK (TGC19). This isdepicted on Slide 7. This construct expresses IFN alpha 2 with a signalsequence for secretion. This vector also expresses the kanamycinresistance protein (translated from the internal IRES element) andallows for selection in transgenic plants. Both TGC18 and TGC19 expresstwo genes as a single transcript, eliminating the need for a secondpromoter, and thus reducing the overall size of each vector. This isimportant as decreasing the overall size of transfected constructsincreases the efficiency with which these elements are able totranslocate to the nucleus, leading to stable integration and theselection of transgenic plants. A single promoter system also reducesthe risk of disrupting flanking gene expression or other situations thatmight ultimately effect transgene expression.

The above listed plasmids may substitute other genes of interest for theinterferon. As will be recognized by those skilled in the art, these maybe substituted at the same locus or at other locations in the plasmids.

A prothrombin encoding sequence (SEQ. ID. NO. 36) may also be integratedinto a plasmid vector in accordance with aspects of the presentinventions as diagrammatically illustrated in FIG. 8. Prothrombin is theprecursor protein to thrombin. It is cleaved at 2 sites by activatedFactor X to release activated thrombin, a coagulation protein whichconverts soluble fibrinogen into insoluble strands of fibrin. Theprothrombin gene cassette (1874 base pairs) was amplified using PCRprimers (Table I for primer sequence). The eGFP gene cassette wasreleased from pzUSEIrK (TGC18) vector by the restriction enzyme BamHI.The resulting 5′ overhangs were subsequently removed with Mung Beannuclease to create blunt ends. The blunt ends were dephosphorylatedusing calf intestinal phosphatase (CIP) by incubating at 50 degreesCelsius for 2 hrs, before ligating overnight with the prothrombin genecassette at 15 degrees Celsius. This created pzUSTIrK (TGC20) (SEQ. ID.NO. 42). PCR generated clones were sequenced to confirm the absence ofany DNA mutations.

A Dermcidin (DCD) encoding sequence (SEQ. ID NO. 30) may also beintegrated into a plasmid vector in accordance with aspects of thepresent inventions as also diagrammatically illustrated in FIG. 8.Dermcidin was a recently reported broad spectrum antimicrobial peptidefound constitutively expressed in the sweat glands. This protein issecreted into the sweat and transported to the epidermal surface. ThePCR amplified DCD gene cassette has flanking BamHI restriction enzymesites (Table I for primer sequence). The BamHI digested DCD genecassette was ligated into the BamHI site of pzUSEIrK (TGC18) vector thatused to be occupied by eGFP, creating pzUSDIrK (TGC21) vector (SEQ. ID.NO. 40). PCR generated clones were sequenced to confirm the absence ofany DNA mutations.

A human Growth Hormone (hGH) encoding sequence (SEQ. ID. NO. 32) mayalso be integrated into a plasmid vector in accordance with aspects ofthe present inventions as also diagrammatically illustrated in FIG. 8.The hGH gene cassette was created by PCR amplification with flankingBamHI restriction enzyme sites (Table I for primer sequence). Then theBamHI digested hGH gene cassette was ligated into the BamHI site ofpzUSEIrK (TGC18) vector that used to be occupied by eGFP, creatingpzUSHIrK (TGC22) vector (SEQ. ID. NO. 41). PCR generated clones weresequenced to confirm the absence of any DNA mutations.

A human interferon gamma (hIFNg) encoding sequence may also beintegrated into a plasmid vector in accordance with aspects of thepresent inventions as also diagrammatically illustrated in FIG. 8. ThehIFNg gene cassette was created by PCR amplification with flanking BamHIretrsction enzyme sites (Table I for primer sequence). The BamHIdigested hIFNg gene cassette was then ligated into the BamHI site ofpzUSEIrK (TGC18) vector that used to be occupied by eGFP, creatingpzUSIfgIrK (TGC23) vector (SEQ. ID. NO. 37). PCR generated clones weresequenced to confirm the absence of any DNA mutations.

TABLE I  List of primer sequences used to create other expressed genesPrimer name Primer sequence Sequence I.D. prothrombinFAAACCATGGCGCACGTCCGAGGC SEQ. ID. NO. 1 prothrombinRCTACTCTCCAAACTGATCAATGA SEQ. ID. NO. 2 dermcidinFGGGGGATCCACCATGAGGTTCATGACTCTC SEQ. ID. NO. 3 dermcidinRGGCGGATCCCTATAGTACTGAGTCAAGG SEQ. ID. NO. 4 hghFGGGGGATCCACCATGGCTACAGGCTCCCGG SEQ. ID. NO. 5 hghRGGCGGATCCCTAGAAGCCACAGCTGCCC SEQ. ID. NO. 6 hifngFGGGGGATCCACCATGAAATATACAAGTTAT SEQ. ID. NO. 7 hifngRTCCGGATCCTTAATAAATAGATTTAGA SEQ. ID. NO. 8 IFN pst-bamCAACTGCAGGATCCAACAATGGCCTTGA SEQ. ID. NO. 9 CCTTTGCTTTAC IFN sac-xhoCAACTCGAGCTCATTCCTTACTTCTAAAC SEQ. ID. NO. 10 TTTCTTG eGFP bam [noATAGGATCCGTGAGCAAGGGCGAGGAGC SEQ. ID. NO. 11 start codon] TGTTCeGFP sac [no TATGAGCTCTTACTTGTACAGCTCGTCCATGCC SEQ. ID. NO. 12start codon] eGFP f bam [no CAGGGATCCACCATGGTGAGCAAGGGCG SEQ. ID. NO. 13stop codon] AGG eGFP r xho [no AGTCTCGAGCTTGTACAGCTCGTCCATGCSEQ. ID. NO. 14 stop codon] Signal sequenceTATCTGCAGACCATGGTGAACAAACACTT SEQ. ID. NO. 15 AA pst bamCTTGTCCCTTTCGGTCCTCATCGTCCTCCT TGGCCTCTCCTCCAACTTGACAGCCGGGG GATCCSignal sequence GTGAATTCGGATCCCCCGGCTGTCAA SEQ. ID. NO. 16 AA ss-pcrIRES sac ATTGAGCTCAAGCTTCGAATTCTGCAG SEQ. ID. NO. 17 IRES xhoATACTCGAGGTGGCCATATTATCATCGTG SEQ. ID. NO. 18 TTTTTC kan xhoATACTCGAGACCATGATTGAACAAGATG SEQ. ID. NO. 19 GATTGCAC Kan xbaATTTCTAGACCAGAGCCGCCGCCAGCATT SEQ. ID. NO. 20 GACAGG prothrombinFAAACCATGGCGCACGTCCGAGGC SEQ. ID. NO. 21 prothrombinRCTACTCTCCAAACTGATCAATGA SEQ. ID. NO. 22 dermcidinFGGGGGATCCACCATGAGGTTCATGACTCTC SEQ. ID. NO. 23 dermcidinRGGCGGATCCCTATAGTACTGAGTCAAGG SEQ. ID. NO. 24 hhghFGGGGGATCCACCATGGCTACAGGCTCCCGG SEQ. ID. NO. 25 hghRGGCGGATCCCTAGAAGCCACAGCTGCCC SEQ. ID. NO. 26 hifngFGGGGGATCCACCATGAAATATACAAGTTAT SEQ. ID. NO. 27 hifngRTCCGGATCCTTAATAAATAGATTTAGA SEQ. ID. NO. 28These sequences may contain their own signal sequence. Similar to thesequence in alpha interferon which was demonstrated as operable in analoe plant, the native signal sequences within prothrombin, Dermcidin(DCD), human Growth Hormone (hGH), and human interferon gamma (hIFNg)may also be demonstrated to be functional in an aloe plant. Regardless,these protein encoding sequences may be cloned into a vector containingthe alpha amylase signal sequence as illustrated examples of FIG. 8.

In the development of the above reference constructs, all vectors usedfor transformation were grown in 250 ml LB 50 ug/L zeocin and isolatedusing the CsCl method. The vectors were then used in transienttransfection studies to ensure proper expression from each construct.Transfection using the gene gun was performed in maize, tobacco, andaloe, and expression monitored visually for expression of eGFP or byrt-PCR for expression of either IFN or kan.

Transformation

After the DNA constructs including the gene of interest and functionalvarious function units incorporated into vectors are generated, the DNAconstructs are introduced into the aloe cells using a number oftechniques that will be recognized by those skilled in the art uponreview of the present disclosure. These DNA constructs are generallydesigned to promote the formation of stably transformed aloe plants 10.Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present inventions. Some methodsfor incorporation of DNA constructs contained in vectors into aloe cellsor tissues to create stable aloe transformants can include infectionwith A. tumefaciens or A. rhizogenes, infection with replicationdeficient viruses, biolistic transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos, or similar methods. Currently, two of the morecommonly used methods for plant transformation areAgrobacterium-mediated transformation and biolistic transformation.Transformed aloe cells or callus tissues are typically grown inappropriate nutrient medium to select for transformed cells. Frequently,a selection medium includes a toxin or other selecting factor that killsnon-transformed cells. Various medium changes will allow the productionof aloe plants 10 that contain the gene of interest as will berecognized by those skilled in the art upon review of the presentdisclosure.

In the practice of transformation DNA is typically introduced into onlya small percentage of target plant cells in any one transformationexperiment. Marker genes are used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theherbicides to which plants of this inventions may be resistant areuseful agents for selective markers. Potentially transformed cells areexposed to the selective agent. In the population of surviving cellswill be those cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin and paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar orpat) and glyphosate(aroA or EPSPS). Examples of such selectable are illustrated in U.S.Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of whichare incorporated herein by reference. Selectable markers which providean ability to visually identify transformants can also be employed, forexample, a gene expressing a colored or fluorescent protein such as aluciferase or green fluorescent protein (GFP) or a gene expressing abeta-glucuronidase or uidA gene (GUS) for which various chromogenicsubstrates are known.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, for example to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function implants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695, both are incorporated herein by reference and arediscussed in more detail below.

For Agrobacterium tumefaciens based plant transformation system,additional elements present on transformation constructs will includeT-DNA left and right border sequences to facilitate incorporation of therecombinant polynucleotide into the plant genome. With infection withagrobacterium (A. tumefaciens), aloe callus tissue is incubated for 1hour at room temperature with an overnight culture of agrobacteriumincluding the vector incorporating the DNA construct. The Agrobacteriumis grown in appropriate selection medium. The selection medium typicallyincludes streptomycin and kanamycin. The selection medium may alsocontain acetosyringone. The acetosyringone at a concentration of 50 uMto 250 uM typically increases the efficiency of monocot infectivity.After growth on the selection media, infected aloe tissue may betransferred to media in Petri dishes with no selection for two (2) daysin the dark 25 degrees Celsius. An exemplary suitable medium could be MSmedia with acetosyringone. Cefotaxime may then be added for two (2) daysto kill the agrobacterium. Cells are then replated on media containing50 mg/L Kanamycin to select for transformed aloe cells and shootinducing growth factors (0.2mg/L NAA, 2 mg/L BAP) for four (4) weeks, 16hrs light. Regenerating shoots may then transferred to rooting medium (½MS with 0.2 mg/L NAA) to develop roots (after about 4 to 6 weeks) beforebeing transferred to soil.

With biolistic transformation, aloe cells or callus tissue are bombardeddirectly with the vector construct. Various embodiments and aspects ofbiolistic transformation are disclosed in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); V5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat.No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) andAgrobacterium-mediated transformation is described in U.S. Pat. No.5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No.5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of whichare incorporated herein by reference. Further, a variety of biolistictransformation apparatus may be use such as for example a BiolisticPDS-1000/He particle delivery system from Bio-Rad, Inc. In thisapproach, the vector and associated DNA construct may be bound to goldor other particles and fired directly into the aloe cells. The bombardedaloe cells are grown without selection for 4 days in the dark beforebeing transferred to selection media. Again, the selection media mayinclude 50 mg/L Kanamycin. Transformants typically begin to form within8-12 weeks and can be transferred to shooting and rooting media for afurther 8-12 weeks. Once roots have begun to form, plantlets can betransferred to soil.

Transformation can also be achieved using a variety of other techniques.Such techniques include viral infection using replication compromisedviral vector systems, electroporation particularly of callus cells orprotoplasts grown in liquid culture, and PEG or lipid mediated transferinto protoplasts. The selection for transformants could then follow thesame basic steps as that outlined for biolistic transformation.

Once introduced into the aloe callus tissues, constructs may beincorporated into the plant genetic material as is generally illustratedin FIG. 3. In other aspects, the constructs may be stably integratedinto the cell outside of the Aloe plants l Ogenetic material. Dependingon the construct, the protein of interest may or may not be expressedabsent some form of induction of transcription. In one aspect,constructs once introduced into Aloe cells may direct protein synthesisor transport to specific tissues of the Aloe plant 10. Typically, thisoccurs when a targeting signal sequence is included on the protein ofinterest.

Site Specific Integration (Cre-Lox)

In one aspect, the present inventions may utilize site-specificintegration or excision of DNA constructs introduced into an aloe plant.An advantage of site-specific integration or excision is that it can beused to overcome problems associated with conventional transformationtechniques, in which transformation constructs typically randomlyintegrate into a host genome in multiple copies. This random insertionof introduced DNA into the genome of host cells can be lethal if theforeign DNA inserts into an essential gene. In addition, the expressionof a transgene may be influenced by “position effects” caused by thesurrounding genomic DNA. Further, because of difficulties associatedwith plants possessing multiple transgene copies, including genesilencing, recombination and unpredictable inheritance, it is typicallydesirable to control the copy number of the DNA constructs inserted intothe transgenic aloe plant's 10 genome, often only desiring the insertionof a single copy of the DNA construct.

Site-specific integration or excision of transgenes or parts oftransgenes can be achieved in plants by means of homologousrecombination (see, for example, U.S. Pat. No. 5,527,695, specificallyincorporated herein by reference in its entirety). Homologousrecombination is a reaction between any pair of DNA sequences having asimilar sequence of nucleotides, where the two sequences interact(recombine) to form a new recombinant DNA species. The frequency ofhomologous recombination increases as the length of the sharednucleotide DNA sequences increases, and is higher with linearizedplasmid molecules than with circularized plasmid molecules. Homologousrecombination can occur between two DNA sequences that are less thanidentical, but the recombination frequency declines as the divergencebetween the two sequences increases.

Introduced DNA sequences can be targeted via homologous recombination bylinking a DNA molecule of interest to sequences sharing homology withendogenous sequences of the host cell. Once the DNA enters the aloecell, the two homologous sequences can interact to insert the introducedDNA at the site where the homologous genomic DNA sequences were located.Therefore, the choice of homologous sequences contained on theintroduced DNA will determine the site where the introduced DNA isintegrated via homologous recombination. For example, if the DNAsequence of interest is linked to DNA sequences sharing homology to asingle copy gene of a host aloe cell, the DNA sequence of interest willbe inserted via homologous recombination at only that single specificsite. However, if the DNA sequence of interest is linked to DNAsequences sharing homology to a multicopy gene of the host eukaryoticcell, then the DNA sequence of interest can be inserted via homologousrecombination at each of the specific sites where a copy of the gene islocated.

DNA can be inserted into the host genome by a homologous recombinationreaction involving either a single reciprocal recombination (resultingin the insertion of the entire length of the introduced DNA) or througha double reciprocal recombination (resulting in the insertion of onlythe DNA located between the two recombination events). For example, ifone wishes to insert a foreign gene into the genomic site where aselected gene is located, the introduced DNA should contain sequenceshomologous to the selected gene. A single homologous recombination eventwould then result in the entire introduced DNA sequence being insertedinto the selected gene. Alternatively, a double recombination event canbe achieved by flanking each end of the DNA sequence of interest (thesequence intended to be inserted into the genome) with DNA sequenceshomologous to the selected gene. A homologous recombination eventinvolving each of the homologous flanking regions will result in theinsertion of the foreign DNA. Thus, only those DNA sequences locatedbetween the two regions sharing genomic homology become integrated intothe genome.

Although introduced DNA sequences can be targeted for insertion into aspecific genomic site via homologous recombination, in higher eukaryoteshomologous recombination is a relatively rare event compared to randominsertion events. In aloe cells, foreign DNA molecules find homologoussequences in the aloe plant's genome and recombine at a frequency ofapproximately 0.5-4.2 times 10⁻⁴. Thus any transformed cell thatcontains an introduced DNA sequence integrated via homologousrecombination will also likely contain numerous copies of randomlyintegrated introduced DNA sequences. One way of avoiding these randominsertions is to utilize a site-specific recombinase system. In general,a site specific recombinase system consists of three elements: two pairsof DNA sequence (the site-specific recombination sequences) and aspecific enzyme (the site-specific recombinase). The site-specificrecombinase will catalyze a recombination reaction only between twosite-specific recombination sequences.

A number of different site specific recombinase systems could beemployed in accordance with the instant inventions, including, but notlimited to, the Cre/lox system of bacteriophage P1 (U.S. Pat. No.5,658,772, specifically incorporated herein by reference in itsentirety), the FLP/FRT system of yeast (Golic and Lindquist, 1989), theGin recombinase of phage Mu (Maeser and Kahmann, 1991), the Pinrecombinase of E. coli (Enomoto et al., 1983), and the R/RS system ofthe pSR1 plasmid (Araki et al., 1992). The bacteriophage P1 Cre/lox andthe yeast FLP/FRT systems constitute two particularly useful systems forsite specific integration or excision of transgenes. In these systems, arecombinase (Cre or FLP) will interact specifically with its respectivesite-specific recombination sequence (lox or FRT, respectively) toinvert or excise the intervening sequences. The sequence for each ofthese two systems is relatively short (34 by for lox and 47 bp for FRT)and therefore, convenient for use with transformation vectors.

FIG. 8 illustrates a particular exemplary protocol using a Flp/Frt andCre/Loxp recombinase system. In such a system, site-specificrecombinases from bacteriophage and yeasts may be used as tools tomanipulate DNA both in the test-tube and in living organisms.Recombinases/recombination site combinations include Cre-Lox, FLP-FRT,and R-RS, where Cre, FLP and R are recombinases, and Lox, FRT, and RSare the recombination sites. To use this system, a transgenic plantcontaining the specific sites for recombination is generated. A parenttransgenic aloe plant 10 is created by transfecting a vector, expressinga selectable marker and engineered to contain Frt and Lox sites intandem. Both the Frt and Lox sites consist of three elements, a spacersequence of eight (8) nucleotides between two (2) inverted repeats ofthirteen (13) nucleotides each. The inverted repeats serve as the DNAbinding domain for the specific recombinase while the spacer element isvariable but essential for homologous recombination. By altering thespacer element of either the LoxP or FRT sites, multiple distinct sitesfor recombination can be created. By alternating Frt and Lox sites asystem allowing multiple site directed integrations can be created (asoutlined in FIG. 8). This system may have a number of advantages.Disruption of endogenous genes is minimized after the generation of theinitial parent plant. The efficiency of transformation is increased bythe expression of site specific recombinases. Using the lox and frtsites in tandem allows for the selective removal of selection markerswhile retaining the gene of interest. And once a parent plant has beencreated cellular propagation and regenerative potential is enhanced. Theparent plant however is the first step and is created following standardtransformation methods (biolistics or Agrobacterium). Subsequenttransgenic plants are created by co-transfection of a vector containingboth a LoxP and Frt site as well as the gene of interest and aselectable marker, together with a vector expressing the Crerecombinase. Expression of the Cre recombinase induces homologousrecombination through the LoxP sites of the vector and the parent plant.Transformants are selected by expression of selectable markers andinduced to regenerate. Subsequent transformation with a vectorexpressing Flp recombinase can remove the selectable marker and allowfor subsequent integration into other Loxp sites.

Production of Viable Plant

Following transformation of Aloe cells or Aloe tissues, the transformedAloe cells or Aloe tissues may be grown into plantlets. Aloe cells thatsurvive exposure to the selective agent, or aloe cells that have beenscored positive in a screening assay, may be cultured in regenerationmedia and allowed to mature into transgenic aloe plants 10. Developingaloe plantlets regenerated from transformed aloe cells can betransferred to plant growth mix, and hardened off, for example, in anenvironmentally controlled chamber at about 85% relative humidity, 600ppm CO₂, and 25-250 microeinsteins m⁻²s⁻¹ of light, prior to transfer toa greenhouse or growth chamber for maturation. The transformed aloeplants 10 are regenerated from about 6 weeks to 10 months after atransformant is identified, depending on the initial tissue. Theregenerated transformed aloe plant 10 or its progeny seed or plants cantypically be tested for expression of the recombinant DNA.

Regenerating a transgenic aloe plant 10 from transformedundifferentiated callus tissue or meristem tissue involves primarilymanipulating the growth factors auxin and kinetin. The first step is topromote shoot growth. Shoot inducing medium typically contains a lowconcentration of auxin (0.2 mg/L NAA) and a relatively highconcentration of cytokinin (2 mg/L BAP). Reduced sugar concentration to2% sucrose (which affects the osmotic pressure) can also help in plantregeneration. Cells on shooting medium are placed in incubators at 25°C. with a 12 hr/day light cycle. Shoots that begin to form are excisedfrom the primary callus after they reach a length of roughly 2 cm andplaced in rooting medium. These shoots can also be maintained inshooting medium to encourage the development of further shoots. Rootingmedium promotes root development and typeically contains ½ MS medium anda relatively low concentration of auxin (0.2 mg/L NAA). After rootsbegin to develop (1-2 cm) the plantlets can be transferred to soil andacclimatized by gradually reducing the relative humidity. Oncegenerated, the transgenic aloe plants  may be propagated by seed, clonalpropagation methods, or otherwise as will be recognized by those skilledin the art upon review of the present disclosure.

Extraction and Purification of Proteins from Plants or Plant Cells

The structural proteins or enzymes introduced via vectors into plantcells and plants may be found in various tissues of the Aloe plant 10including roots, tubers, leaves, seeds, flowers, sap or in a combinationof plant parts and developmental stages. In one aspect, the protein orproteins of interests are concentrated in the gelatinous matrix in thecentral portion of the leaf. In another aspect, the protein or proteinsof interest are biologically active and provide some degree of medicinaleffect when extracted from the Aloe leaf 12. In another aspect, theprotein or proteins of interest and the at least one native aloeprotein, carbohydrate and/or other compound found of the gelatinousmatrix act synergistically to provide a desired treatment for a patient.Protein extraction can be from total biomass or a particular tissue.Protein extraction from plant cells may include physical and chemicalmethods. Protein extraction from leaves or sap may involve filtration,ultracentrifugation, chemical extraction and affinity chromatography.

In one aspect, the protein of interest may accumulate within the gel ofthe leaf of the transformed aloe plant 10. This region is primarilywater, roughly 99%, and free of harmful proteases. Extraction of the gelis an established technique that will be understood by those skilled inthe art. Some of the proteins, particularly cosmetic, may be used tosupplement the aloe gel directly and may never need to be individuallyisolated from the gel.

EXAMPLES Methodologies for Isolation of Plant Cells

In a first example, shoot meristem tissue for direct use or for callusinitiation is isolated from the stalk of the aloe, either Aloe vera,Aloe ferox or Aloe arborescence.

The leaf sections are removed and the stalk is cut laterally through thelongitudinal axis of the aloe leaf. Tissue is sterilized with a mixtureof Tween 20 (0.05%) and hypochlorite (5%) for 10 min, followed by 30seconds ethanol, rinse 3× sterile water. Sections are then plated withthe exposed surface lying sideways to the plate in MS media with agar(0.8-1%) containing various concentrations of the growth factors, auxinand cytokinin, and 2-3% sucrose. Growth conditions vary depending on thedesired process. Callus culture without shoot development is typicallyinitiated by growing these sections in NAA (2-5 mg/L) without BAP orwith low concentration of BAP (0.2 mg/L). Undifferentiated cells beginto grow along the areas of the cut stalk and can be subcultured andmaintained on separate dishes. Shoot induction can be initiated directlyby placing such fragments in shooting medium—MS media containing 0.2mg/L NAA (or IAA 0.2 mg/L) and 2 mg/L BAP. Both NAA and IAA work as asource of auxin and there is a range of concentrations of both auxinsand cytokinins that have effect. Callus cells from immature embryos aredeveloped from commercially available seeds. The seeds are firststerilized (ethanol 30sec, 5% hypochlorite plus 0.05% Tween 20 for 15minand rinsed 3× in sterile water). Sterilized seeds are then leftovernight at 4 degrees Celsius in water. The immature embryo is isolatedby removing the seed coat. A small cut is made at one corner of thetriangular shaped seed and the embryo is squeezed out. This is thenplated on MS media containing either auxin alone (NAA 2-5 mg/L) toinduce callus, or auxin and cytokinin (NAA 0.2 mg/L, BAP 2 mg/L) toinduce callus and shoot propagation. Sugar concentration is typically2-3%.

Callus cells from the shoot apical meristem are developed from a one totwo centimeter segment cut from the base of the young aloe plant 10 withall the leaves removed. The fragment is surface sterilized and replatedin 1% agar with MS media and auxin and cytokinin (2 mg/L NAA 0.2 mg/LBAP).

Callus cell induction initiates within 1-2 weeks. Cells can then beisolated from these cultures and used as starting material fortransformation.

DNA Constructs

i. pBI121 Ubiquitin Interferon Vector (pBI-UI).

The pBI121 Agrobacterium binary vector was used as the backbone for thecreation of pBI-UI. The human interferon alpha 2 gene with signalsequence was amplified from human 293 cells using gene specific primerscontaining 5′ PstI and 3′ SacI/XhoI restriction sites. The 587 byinterferon PCR product was cloned into pZErO and sequenced. The 1962 byubiquitin (Ubi) promoter element from maize was also cloned into pZErOby amplifying the region from the vector pUBI-GFP and cloning into 5′HindIII and 3′ PstI restriction sites. The Ubi fragment was thensubcloned into the pZero interferon vector using HindIII/PstI (pZErOUI). The intact Ubi interferon cassette was then subcloned into pBI121using HindIII/SacI and removing the CaMV 35S promoter and GUS generesulting in pBI UI. This vector retains the right and left T-elementborder regions necessary for agrobacterium mediated infection andtransformation and expresses the kanamycin resistance gene (NPTII) underthe control of the Nos promoter.

ii. pZErO Ubiquitin Interferon IRES Kanamycin. (pZ UIIK).

This vector was created by cloning the Ubi promoter into the pZErOcloning vector. Behind this fragment was cloned a cassette containinghuman interferon alpha 2 together with an IRES (internal ribosomal entrysequence) and the kanamycin resistance gene. This unit is expressed as asingle transcript but translated as two distinct proteins (due to thesecond translational initiation site provided by the IRES). This allowsboth the selectable marker and interferon to be expressed under thecontrol of the Ubi promoter.

iii. The Double Plasmid System Employing Cre and Flp Eecombinases.

A parent plant is first generated which expresses the selectable markerhaving the cre and flp sites for targeted integration. A secondarytransfection introducing vectors with genes of interest is targeted tothe cre sites. Unwanted genetic material is then removed using flp.

Introduction of DNA Constructs into Isolated Plant Cells

i. Agrobacterium Mediated Gene Transfer.

The Agrobacterium strain LB4404 (Invitrogen, Carlsbad, Calif.) waselectroporated with the pBI UI vector and positive clones selected on LBagar plates containing 50 ug/ml kanamycin and 100 ug/ml streptomycin.Individual clones were grown overnight at 30 degrees Celsius in LB mediacontaining 250 uM acetosyringone. Infection took place by submergingplant fragments or callus in the overnight cultures, blotting dry onsterile filter paper, and plating on MS agar plates containing 250 uMacetosyringone at 25 degrees Celsius in the dark. Two days afterinfection, tissue was transferred to MS agar plates containing 200 uMcefotaxime to kill the Agrobacterium. The tissue is then transferred toMS agar plates containing 50 mg/L kanamycin and 0.2 mg/L NAA and 2 mg/LBAP to select for transformants and induce shoot regeneration. Thetissue is grown in 12 hours light at 25 degrees Celsius. Adventitiousshoots are grown until they reach roughly 2 cm in length before they areexcised and replanted on rooting media with continued selection.Plantlets expressing genes of interest are assayed for expression levelsand transferred to soil.

ii. Biolistic Gene Transfer.

Gold particles (0.6-1 um) are coated with the vector including the DNAconstruct. The gold particles are washed 15 minutes in 70% ethanol byvortexing and soaking, followed by 3 washes in sterile water. The goldparticles are then resuspended in 50% glycerol to a concentration of 60mg/ml. To coat vector DNA on the washed particles, 3 mg of particles areadded to a 1.5 ml microfuge tube. To this is added 5 ul DNA at aconcentration of 1 ug/ul, 50 ul 2.5M CaCl2, and 20 ul 0.1 M spermidine,and vortexed for 2-3 min. This is allowed to settle, spun for 1-2 secand the liquid removed. To this is added 140 ul of 70% ethanol, spun,and the liquid discarded. To this is added 140 ul 100% ethanol, spun andthe liquid discarded. To the precipitate is added 48 ul 100% ethanol andgently resuspended. The gene gun apparatus (PDS 1000/He, Bio-RadLaboratories, Inc., Hercules, Calif.) is sterilized using 70% ethanol.The gene gun apparatus was assembled using the shortest gap distancebetween the macrocarrier holder and stopping screen (recommendedsetting). Coated gold particles (8 ul) are pipetted onto the center ofthe macrocarriers. The target distance was set depending upon the targettissue (6 cm tissue, 9 cm callus) with a rupture disk of 600-1100 psi.

In a first example, following bombardment cells were plated on MS agarplates containing 2 mg/L NAA without selection for 1 week in the dark at25 degrees Celsius. Cells were then transferred to MS agar platescontaining 50 mg/L kanamycin for selection and auxin (2 mg/L NAA) andcytokinin (0.2 mg/L, 6-benzylaminopurine) for 4-5 more weeks. The cellsare grown for 12 hours in light at 25 degrees Celsius.

In a second example, microprojectile bombardment was carried out on aplate of MS (4 g/L MS salts, 1 mg/L (1000×) MS vitamin stock, 2 mg/LNAA, 100 mg/L myo-inositol, 2.76 g/L proline, 30 g/L sucrose, 100 mg/Lcasein hydrolysate, 36.4 g/L sorbitol, 36.4 g/L mannitol, 2.5 g/Lgelrite, pH 5.8). The bombarded callus was left on MSOSM for 1 hourafter bombardment and then transferred to MS initiation medium (4 g/L MSsalts, 1 mg/L (1000×) MS vitamin stock, 2 mg/L NAA, 100 mg/Lmyo-inositol, 2 g/L proline, 30 g/L sucrose, 100 mg/L caseinhydrolysate, 2.5 g/L gelrite, pH 5.8). After 7-10 days on MS, thebombarded callus was transferred to MSS selection medium (4 g/L MSsalts, 1 mg/L (1000×) MS vitamin stock, 2 mg/L NAA, 100 mg/Lmyo-inositol, 30 g/L sucrose, 2.5 g/L gelrite, pH 5.8) with 50 mg/Lkanamycin for selection of transformed cells. After 3 weeks, individualcallus pieces were transferred to fresh MSS medium. Within 6-8 weeks ofbombardment, kanamycin resistant clones emerged from selected calluspieces.

iii. Protoplast.

Protoplasts are plant cells lacking their outer cell wall. The advantageof creating these cells is to increase the efficiency of transfectionand such cells are able to fuse together forming a somatic cell hybrid.Somatic cell hybrids could mix up the genes from different plantspotentially arriving at something completely new.

The technique for using protoplasts in accordance with the presentinventions may include growing the cells in liquid culture (2-3 days inlogarithmic growth phase). The cells are then spun down (1000×Gravityfor 5 min). The cells are resuspended in a solution containingcellulase, macerozyme, and pectolyase. Shake over night at 25 degreesCelsius in the dark. Centrifuge (600×Gravity for 5 min.) and remove thesupernatant. The protoplasts are resuspended in culture medium and spunone more time to remove traces of enzymes. The protoplasts cannot bestored or propagated and must be used for transfection or somatic cellfusion relatively quickly.

Production of Viable Plants Containing DNA Constructs

In a first example, successful transformants are selected initially fortheir resistance to cellular toxins, e.g. kanamycin. Stably transfectedcells must express both the resistance marker and the gene of interest,such as for example a gene encoding interferon, as well to be able toregenerate the intact plant. 50 mg/L kanamycin is added to the MS agarplates to initiate selection. The course of selection depends in part onthe speed with which the cells replicate but can take between 6 to 10weeks. During this time transformed tissue is induced to regenerate byadding 0.2 mg/L NAA and 2 mg/L BAP to the MS agar plates and encouragingshoot development with a 12 hr/day light cycle (with continued selectionpressure). As adventitious shoots begin to develop they can be assayedfor gene expression at about 2 cm in length. Shoots expressing desiredgene products (as assayed by RT-PCR, Western blot, and biological assay)are subsequently transferred to MS agar plates to induce root formation(½ MS plus 0.2 mg/L NAA).

In a second example, regeneration of transgenic callus is accomplishedby transferring about 15 small pieces (approximately 4 mm) toRegeneration Medium I (4.3 g/L MS salts, 1 ml/L (1000×) MS vitaminstock, 100 mg/L myo-inositol, 60 g/L sucrose, 3 g/L gelrite, pH 5.8)with added filter sterilized kanamycin (50 mg/L) and incubating for 2-3weeks at 25 degrees Celsius in the dark. Matured somatic embryos aretransferred to the light on Regeneration Medium II for germination (4.3g/L MS salts, 1 ml/L (1000×) MS vitamin stock, 100 mg/L myo-inositol, 30g/L sucrose, 3 g/L gelrite, pH 5.8) with added filter sterilizedkanamycin (50 mg/L).

What is claimed is:
 1. A transgenic aloe plant comprising a recombinantDNA construct, said construct comprising a promoter, a sequence encodingan exogenous protein, a termination sequence, and a translocationsequence encoding a secretion signal peptide, wherein the promoter is anubiquitin promoter from maize having SEQ ID NO. 44; and wherein theexogenous protein is expressed from the DNA construct.
 2. The transgenicaloe plant of claim 1, wherein at least a portion of the exogenousprotein is translocated from an aloe cell into the gel of an aloe leafof the transgenic aloe plant
 3. The transgenic aloe plant of claim 1,wherein the exogenous protein is a mammalian protein selected frominterferons, immunoglobulins, mammalian growth factors, mammalianhormones, blood factors, and histocompatibility antigens.
 4. Thetransgenic aloe plant of claim 1, wherein the exogenous protein is amammalian protein selected from a-interferon, y-interferon, prothrombin,dermicidin and human growth hormone.
 5. The transgenic aloe plant ofclaim 1, wherein the translocation sequence is the alpha amylasesecretory sequence from rice.
 6. The transgenic aloe plant of claim 5,wherein the translocation sequence is the alpha amylase secretorysequence from Oryza sativa rice having SEQ ID No.
 29. 7. The transgenicaloe plant of claim 1, wherein the recombinant DNA construct has atleast one enhancer element, at least one selectable marker gene, or acombination thereof.
 8. The transgenic aloe plant of claim 1, whereinthe recombinant DNA construct has at least one selectable marker geneconferring resistance to a compound chosen from the group consisting ofkanamycin, chloramphenicol, G418, hygromycin B, paromomycin,glufosinate, glyphosate and gentamycin.
 9. The transgenic aloe plant ofclaim 1, wherein the recombinant DNA construct further comprises anexpression cassette encoding eGFP, the expression cassette encoding eGFPhaving an internal ribosomal entry site and a termination sequence,wherein both the sequence encoding the exogenous protein and eGFPsequences are expressed as a single mRNA transcript.
 10. The transgenicaloe plant of claim 1, wherein the recombinant DNA construct furthercomprises an expression cassette encoding a protein conferringresistance to kanamycin, the sequence encoding the protein conferringresistance to kanamycin having an internal ribosomal entry site and atermination sequence, wherein both the sequence encoding the exogenousprotein and the kanamycin-resistant sequences are expressed as a singlemRNA transcript.
 11. A method for producing an exogenous protein in anAloe plant, the method comprising: providing a transgenic Aloe plantcomprising a recombinant DNA construct comprising a promoter, a sequenceencoding the exogenous protein, a termination sequence and atranslocation sequence encoding a secretion signal peptide; wherein thepromoter is a ubiquitin promoter from maize having SEQ ID No. 44; andcultivating the plant so that the exogenous protein from the DNAconstruct is expressed, wherein at least a portion of the exogenousprotein is translocated from an aloe cell into the gel of a leaf of thetransgenic plant.
 12. The method of claim 11, wherein the exogenousprotein is extracted from the gel of the leaf of the transgenic plant.13. The method of claim 11, wherein the exogenous protein is a mammalianprotein selected from α-interferon, γ-interferon, prothrombin,dermicidin and human growth hormone.
 14. The method of claim 11, whereinthe translocation sequence is the alpha amylase secretory sequence fromrice (Oryza sativa).
 15. The method of claim 11, wherein the transgenicAloe plant is generated by a process comprising: isolating aloe cellsfrom an aloe seed, meristem or plant; growing the aloe cells in culturein a nutrient medium containing an auxin, and a cytokinin to form acallus; transforming the callus with the recombinant DNA construct;selecting for transformed aloe cells containing the recombinant DNAconstruct while growing the callus in a shooting and selection mediumcomprising 0.2 mg/L of 1-naphthaleneacetic acid (NAA) or indole 3-aceticacid (IAA), and 2 mg/L of 6-benzylaminopurine (BAP); growing a shootregenerated in the shooting and selection medium in a rooting mediumcomprising 0.2 mg/L NAA; and when roots have begun to form, transferringthe plantlet to soil.
 16. The method of claim 15, wherein the aloe cellsare embryonic aloe cells from an aloe seed, or meristematic cells froman aloe plant, an aloe shoot meristem, or an aloe root meristem.
 17. Amethod of producing a transgenic Aloe plant that expresses a proteinexogenous to the plant, the method comprising: culturingundifferentiated callus of aloe cells in a nutrient medium containing anauxin, and a cytokinin; introducing into the callus a recombinant DNAconstruct comprising a promoter, a sequence encoding the exogenousprotein, a termination sequence and a translocation sequence encoding asecretion signal peptide; wherein said promoter comprises SEQ ID No. 44;wherein the exogenous protein is expressed from the DNA construct;growing the callus in a shooting and selection medium comprising 0.2mg/L of 1-naphthaleneacetic acid (NAA) or indole 3-acetic acid (IAA),and 2 mg/L of 6-benzylaminopurine (BAP); growing a shoot regenerated inthe shooting and selection medium; and once roots have begun to form,transferring the plantlet to soil.
 18. The method of claim 17, whereinat least a portion of the exogenous protein is translocated from an aloecell into the gel of an aloe leaf of the transgenic aloe plant.
 19. Themethod of claim 17, wherein the exogenous protein is a mammalian proteinselected from α-interferon, γ-interferon, prothrombin, dermicidin andhuman growth hormone.
 20. The method of claim 17, wherein the callus isfrom aloe seed.